title: Combinatorial targeting of 2 different steps in adenoviral DNA replication by herpes simplex virus thymidine kinase and artificial microRNA expression for the inhibition of virus multiplication in the presence of ganciclovir.
abstract: BACKGROUND
  Human adenoviruses are a frequent threat to immunocompromised patients, and disseminated disease is associated with severe morbidity and mortality. Current drugs are not capable of preventing all fatalities, thus indicating the need for alternative treatment strategies. Adenoviruses can be rendered susceptible to antiherpetic prodrugs such as ganciclovir (GCV), upon expression of the herpes simplex virus thymidine kinase (HSV-TK) gene in adenovirus-infected cells. Furthermore, adenoviruses are amenable to post-transcriptional gene silencing via small interfering RNAs (siRNAs) or artificial micro RNAs (amiRNAs).
  
  
  RESULTS
  In this study, we combined these 2 approaches by constructing a combinatorial gene expression cassette that comprises the HSV-TK gene and multiple copies of an amiRNA directed against the mRNA encoding the adenoviral preterminal protein (pTP). HSV-TK gene expression was controlled by the adenoviral E4 promoter, which is activated in the presence of the adenoviral E1 gene products (i.e., when adenovirus is present in the cell). When inserted into a replication-deficient (E1-, E3-deleted) adenoviral vector, this cassette effectively inhibited the replication of wild-type adenovirus in vitro. The reduction rate mediated by the combinatorial approach was higher compared to that achieved by either of the 2 approaches alone, and these obvious additive effects became most pronounced when the GCV concentration was low.
  
  
  CONCLUSIONS
  The concept presented here has the potential to aid in the inhibition of wild-type adenovirus replication. Furthermore, the combinatorial expression cassette may constitute a safeguard to potentially control unintended replication of adenoviral vectors and to prevent immune responses provoked by them.
authors.affiliation: Children's Cancer Research Institute, St, Anna Kinderkrebsforschung, Zimmermannplatz 10, 1090 Vienna, Austria.
authors.forename: Mirza
authors.initials: M
authors.lastname: Ibrišimović
authors.forename: Thomas
authors.initials: T
authors.lastname: Lion
authors.forename: Reinhard
authors.initials: R
authors.lastname: Klein
mesh_terms: D000256:Adenoviridae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D000998:Antiviral Agents / Q000494:pharmacology; D001709:Biotechnology; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D015774:Ganciclovir / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D035683:MicroRNAs; D014764:Viral Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir